Tissue remodeling investigation in varicose veins.

Although the etiology of varicose veins remains unknown, recent studies have focused on endothelial cell integrity and function because the endothelium regulates vessel tone and synthesizes many pro- and anti-inflammatory factors. The aim of this study was to investigate the evidence involving the endothelium in the development of varicose vein disease. In addition, tissue remodeling was investigated in varicose veins to determine the expression of different types of collagen. Tissue specimens of superficial varicose veins and control saphenous vein were used for immunohistochemical and transmission electron microscope (TEM). α-smooth muscle actin, and collagen I, III, IV antibodies were applied for immunohistochemical investigation. Findings of this study showed alterations of the intima, such as focal intimal discontinuity and denudation of endothelium; and the media, such as irregular arrangements of smooth muscle cells and collagen fibres in varicose veins. Our findings showed some changes in terms of distribution of types I, III and IV collagen in the intima and media of varicose vein walls compared with controls. These alterations to the media suggest that the pathological abnormality in varicose veins may be due to the loss of muscle tone as a result of the breakup of its regular structure by the collagen fibres. These findings only described some changes in terms of distribution of these types of collagen in the intima and media of varicose vein walls which may result in venous wall dysfunction in varicosis.

smooth muscle cells in the media of the varicose veins (7). These synthetic smooth muscle cells synthesize larger amounts of the extracellular matrix components and lose the expression of the contractile filaments leading to the thickening of the venous wall and loss of the contractility in the varicose vein (8). However, others believe there is a reduction in the cellularity of the smooth muscle layer with replacement by collagen or a significant increase in collagen content of varicose veins (9,10). In normal veins, the extracellular matrix of medial layer, including collagen and elastic fibres, is produced primarily by smooth muscle cells and in adventitial layer, collagen fibres are synthesized and secreted by the adventitial fibroblasts (11).
Although endothelial cells are able to synthesize basement membrane and interstitial collagen, the principal source of collagen in the vessel wall is the smooth muscle cell. Alteration of smooth muscle cells behaviour from quiescent or "contractile state" typical of the normal vessel phenotype to a proliferative or "synthetic state" characteristic of the atherosclerotic phenotype increases collagen synthesis (12).
It was found that in this situation, type I collagen synthesis increases. Similar collagen changes occur in phenotypically altered smooth muscle cells in hypertension (13). The amount of collagen and elastin in the veins, and especially the saphenous veins, contain more collagen than elastin (47% and 7% of the dry weight for collagen and elastin, respectively) (10). The predominant vascular collagens are types I and III, which comprise up to 80-90% of the total blood vessel wall collagens (Type I collagen about 60% and type III about 30% of the total collagen (14). Type IV collagen is a major component of the basal lamina of blood vessels, which plays an important role in regulating pro-and anti-angiogenic events (15). It is distributed in sub-endothelial cells of intima and around smooth muscle cells in the media and in the basement membrane of the vasa vasorum and nerve fibres in the adventitia (16).
Moreover, there is some variation in the amount and location of vascular collagen in different forms of vascular diseases (17). For instance, larger areas and higher amounts of collagen were identified in varicose veins compared to controls (18

Electron Microscopy
For electron microscopy the tissue was washed with PBS and then cut into blocks no bigger than 1mm3 (the vein was cut into small pieces cross-sectionally). Preparation of sections was carried out as described previously (24,25  appeared thicker than the outer layer in some parts of the media (Fig.2a,b). The circular smooth muscle was disorganized and the thickness of its bundles appeared to be reduced in 4 (20%) varicose vein specimens (Table1).    specimens (30%) (Fig.4a,b) ( Table 1).

Distribution of type III collagen in 3 (15%)
varicose vein specimens showed weak staining in the media. In all control saphenous vein, the immunostaining of type III collagen was scattered between smooth muscle cells beneath the endothelial cell layer (Fig.5a).
Immunolocalisation of type III collagen was found in the subendothelial layer of all varicose vein specimens (100%) and in the media and the adventitia of 16 specimens (85%) (Fig.5b) (Table1). Type IV collagen staining of control (Fig.6a) and varicose saphenous (Fig.6b) (Fig.7a). However, thickness of collagen fibres in area where the endothelial cells are lost in varicose veins (Fig.8a) appeared to be greater than in the few areas of endothelial cells loss in control saphenous veins (Fig.8b).  (Fig.9a,b). veins (30,31), whereas, others found reduced amounts of smooth muscle cells due to replacement by connective tissue (6,22).
Immunostaining of the adventitia in this study confirmed previous observations (28)